Despite recent improvements in mortality for breast cancer patients, it is still not known why many patients relapse even after an initial response to conventional endocrine and/or chemotherapies. We have approached this question using both genetically engineered mouse models and in patients in the clinic. Using a unique transplantable p53 null mammary tumor model we have identified a highly enriched Tumor-Initiating Cell (TIC) subpopulation. Both in vitro and in vivo experiments indicate that more efficient DNA damage repair mechanisms exist in these TICs as compared to the bulk of the tumor cells. In parallel studies, an epithelial- mesenchymal transition(EMT)-related gene signature was identified in TICs isolated from breast cancer patients. This signature was present in the subset of human breast cancers characterized as claudin-low, and most importantly was significantly enriched in patients who failed both hormonal and chemotherapy. Often TICs represent a small subpopulation of cells within a tumor, but claudin-low tumors of both mice and humans appear to be highly enriched in TICs. TICs may be intrinsically more resistant to DNA damage induced by radiation and chemotherapy treatments, thus in part explaining their resistance to conventional treatments; they have also been suggested to be responsible for metastatic dissemination, tumor dormancy and recurrence. Thus, to help elucidate the mechanisms and signaling pathways that are responsible for the intrinsic therapeutic resistance of TICs, and to develop therapies that can overcome this resistance to conventional treatments, the following Specific Aims are proposed: Aim 1. Analysis of TICs in the different subtypes of the murine p53 null tumors including the claudin- low/spindloid class. Aim 2. To determine if perturbation of EMT pathways in the p53 null claudin-low/spindloid tumors alters their phenotypic properties. Aim 3. To determine the therapeutic sensitivity of TICs. One pressing need to effectively target breast cancer tumor initiating cells is the development of improved preclinical models to test these therapies. To address this need, we have identified mouse models that develop claudin-low/spindloid tumors, where the bulk of the tumors cells appear to be TICs. Thus, we believe that we have an appropriate and validated model for the investigation of important signaling pathways and therapeutics. This multiPI grant combines the considerable stem cell and signaling pathway expertise in the Rosen laboratory with the genomics, bioinformatics and therapeutics expertise in the Perou laboratory and, therefore represents an excellent synergy between the two. )